1. Field of the Invention
The present invention pertains to the field of analysis and separation of DNA, RNA and proteins. In particular, the present invention refers to an apparatus that allows for the separation and analysis of DNA, RNA and proteins.
2. Description of the Related Art
This invention concerns an apparatus for separating and analyzing DNA, RNA and proteins. Particularly, it relates to a capillary electrophoresis system effective to sequencing of DNA and RNA or, measurement of polymorphism, based on the versatility of individuals.
Analyzing technology for DNA and RNA has become important more and more in medical and biological fields concerning DNA analysis and DNA diagnosis. DNA analyzers at high speed and high throughput have been developed in relation with genome projects.
Capillary electrophoresis is a method of high speed and high resolution since higher electric fields can be applied due to high electric resistance and high efficiency of heat dissipation compared with slab gel electrophoresis. (Anal. Chem. 62, 900 (1999) (Prior art 1)).
As shown in FIG. 16, an electrophoresis system using a single capillary is known (Prior art 2: WO95/21378). The prior art 2 includes the following descriptions regarding FIG. 1. Nucleic acid fragments are electrophoretically separated in a capillary 134 filled with a flowing polymer solution as an electrophoretic separation medium. The temperature of the capillary during electrophoresis is an important parameter that gives an effect on both the extent of denaturation of samples and the separation speed of fragments. An elevated temperature can be used for lowering the viscosity of the separation medium during introduction of the separation medium into the capillary, to shorten the time of filling. A temperature control unit 130 is used for keeping the capillary 134 filled with the electrophoretic separation medium to a predetermined temperature. Since the resolution and the electrophoresis time are determined partially based on the length of the capillary, the temperature control unit contains a portion of the capillary for the length from 30 to 35 cm. The temperature of the capillary is controlled by using an insulated pressure plate and bringing the capillary in contact with a thermally controlled surface.
A sample is introduced into the capillary 134 filled with the separation medium. A sample injection end 142 of the capillary 134 and a cathode 150 are immersed in a sample 158 contained in a sample container 126 disposed to an auto-sampler 166, a sample elution end 138 of the capillary 134 and an anode 146 are immersed in an electrophoretic buffer 154 contained in an electrode vessel 118 and the sample is introduced into the capillary by application of electric field. An injection voltage is applied to the capillary by a power supply unit 122 connected to the anode 146 and the cathode 150. The injection voltage and injection time are controlled by a computer 174. After the injection of the sample into the capillary, electrophoretic separation is conducted.
The auto-sampler 176 changes the position for the sample injection end 142 of the capillary 134 and the cathode 150 from the container 126 containing the sample 158 to a container 170 containing an electrophoretic buffer 182. A voltage is applied to the capillary 124 by the power supply unit 122 connected to the anode 146 and the cathode 150, and sample ingredients pass the capillary by electrophoresis depending on the size thereof
In order to avoid abnormality in fragment phoresis by siphoning of the electrophoresis medium, it is important to keep the liquid levels of the buffer identical between the container 170 and the container 118 during electrophoretic separation.
The fragment is detected after the separation by a detection unit 162. The detector used herein can include (1) a unit for spectrally separating an emission light (such as a grating or a prism), (2) an array of a plurality of detection elements sensitive to irradiation of light (e.g., diode array, CCD, photomultiplier), (3) an excited light source (e.g., incandescent lamp, arc lamp, laser, laser diode) and (4) a spectral array fluorescence detector using an optical system enabling directionation and conditioning for both of excited light and emitted light.
Before electrophoretic separation of the next sample, the electrophoresis medium in the capillary is replaced with a new one. There are described two methods of replacing the inside of the capillary with the new electrophoresis medium. In one method, the electrophoresis medium is replaced by the following procedures. A positive pressure from a pump 104 is applied to a container 108 containing a new electrophoresis medium and the medium is pumped out from the vessel 108 to a tee 111. Since a valve 112 is closed at this instance, the new electrophoresis medium flows mainly to a sample injection end 142. After the capillary is filled with the new electrophoresis medium between the sample injection end 142 and the tee 111, the valve 112 is opened and a new electrophoresis medium flows from the container 108 to a container 118 for containing a buffer. As a result, the capillary is completely filled with the electrophoresis medium between the sample injection end 142 and the sampling elution end 138. Since the length of the capillary between the sample elution end 138 and the tee 111 is shorter than the length of the capillary between the sample injection end 142 and the tee 111, when the valve 112 is opened to apply a pressure to the container 108, the new electrophoresis medium flows mainly to the sample elution end 138. After the capillary is filled with the new electrophoresis medium between the sample injection end 142 and the sample elution end 138, the valve 112 is opened so as to connect the sample injection end 142 and the sample elution end 138 to form a current path between electrodes 146 and 150. The foregoing provides an are explanation for FIG. 1 of the prior art 2.
An electrophoresis system for DNA using a single capillary is supplied as a commercial product from Perkin Elmer Co. (name of product: ABI Prism 310). A high throughput system capable of analyzing a plurality of samples simultaneously, by arranging 96 capillaries into an array is supported as a commercial product from Perkin Elmer Co. (name of product: ABI Prism 3700).
In genome analysis, DNA fragments formed by finely fragmenting large size DNA, at random are read and an original DNA is read by joining the result of reading. As the DNA read length capable of being read by electrophoresis at one time is increased, the efficiency and the speed of the entire analysis also increase.
Accordingly, in the capillary electrophoresis system, a capillary of increased effective length is used so that long DNA can be read. For example, in ABI Prism 310 or ABI Prim 3700, 600 base lengths can be read in about two hours, by electrophoresis under standard conditions (200 V/cm, 50(C), by setting the effective separation length to 50 cm and using a polymer solution POP 6 supplied from Perkin Elmer Co.
As the speed, the throughput and the read length have been increased for the capillary electrophoresis systems, it is expected that the entire base sequencing of the human genome will be completed substantially in 2001. After the base sequences for the entire genome are found, the necessity for reading long DNA will be reduced. It will only be necessary to read specific regions on the genome.
For example, it has now been advanced in a large scale national project to investigate single nucleotide polymorphisms present on the genome, on every group of persons and examine the relationship thereof with diseases or reactivity to chemicals. In the featured medical treatment and new industries, it will be important to relate not only the single nucleotide polymorphism but also specific sequences with diseases or various phenotypes. It is also possible to examine the expressions in each of the organs and each of the states of individual persons, not just with the genome, based on cDNA sequencing. Since a number of samples such as various persons, various diseases and various organs have to be analyzed, it is necessary to outstandingly improve the analysis throughput of the system. In such DNA analyses, reading long base lengths is not necessary, but relatively short base lengths have to be read at high speed, with higher throughput.
However, the constitution of the system for analyzing a great amount of relatively short DNA is not considered in commercial products and an effective separation length as long as 50 cm has been used. When electrophoresis is conducted under standard conditions (200 V/cm, 50xc2x0 C.), using POP 6 as the polymer solution, a plurality of samples can be electrophoresed serially at 65 min cycles to read 200 bases for each sample, which includes periods of 15 min for filling the polymer solution and pre-electrophoresis. When 96 capillaries are used (in ABI Prism 3700), the upper limit for the analysis is 2127 samples in 24 hours.
This invention intends to provide a high performance capillary electrophoresis system capable of conducting capillary electrophoresis, with short effective separation lengths stably capable of electrophoresis at high throughputs and ultrahigh speeds, and applicable to the separation and analysis of nucleic acids and proteins or the sequencing of nucleic acids, and further to measurements for polymorphism analysis, based on the versatility of base sequences of individuals.
The present invention is a capillary electrophoresis system that consists of a capillary for separating fluorescently labeled samples by electrophoresis by applying a voltage across both ends of said capillary, an irradiation unit for irradiating a laser beam to the capillary and a detection unit for detecting fluorescence emitted from a plurality of electrophoretically separated samples. A relationship is maintained between the geometrical characteristics of the capillary: T the height of a highest position of the capillary in a vertical direction, measured from a reference horizontal plane, E1 the height of the sample injection end in vertical direction from the reference horizontal plane, and L1 the capillary length between the sample injection end and the fluorescence detection position at which the laser beam is irradiated. The relationship is Txe2x88x92E1 greater than L1. The sample injection end points substantially vertically downward.
The capillary electrophoresis system further comprises a temperature controller that sets at least a part of the capillary at a selected temperature during electrophoresis. A line connecting the sample injection end and the fluorescence detection position is substantially parallel with a vertical line. Also, a line connecting the sample injection end and the sample elution end of the capillary is substantially parallel with a vertical line.
The capillary electrophoresis system further comprises an electrolyte vessel with a gas permeation membrane, and a tube connected to the gas permeation membrane. A plurality of gases are sucked from an electrolyte set that is part of the electrolyte vessel through the gas permeation membrane. The electrolyte vessel is on one side of the sample elution end and is sealed. A pressure equal or higher that the atmospheric pressure is applied to the electrolyte vessel on the sample injection end. The capillaries used are provided. The sample injection end, the plurality of fluorescence detection positions and the sample elution end are arranged substantially at an identical distances from each other, on an identical plane.
Further, in a different embodiment of the invention, a capillary electrophoresis system comprises a capillary containing a solution of an electrophoretic separation medium for separating fluorescent-labeled samples using electrophoresis created by applying a voltage across both ends of said capillary and an irradiation unit for irradiating a laser beam on to the capillary. The capilarity has a fluorescence detection position at which a laser beam is irradiated. The movements of a plurality of samples placed at the fluorescence detection position are directed vertically upward, while the laser beam is irradiated.
The capillary electrophoresis system further comprises a temperature controller that sets at least a part of the capillary at a selected temperature during electrophoresis. A line connecting a sample injection end and a fluorescence detection position is substantially parallel with a vertical line. Also, a line connecting a sample injection end and a sample elution end of the capillary is substantially parallel with a vertical line.
The capillary electrophoresis system further comprises an electrolyte vessel with a gas permeation membrane and a tube connected to the gas permeation membrane. The gases are sucked from an electrolyte set into the electrolyte vessel through the gas permeation membrane. The electrolyte vessel is on one side of the sample elution end and is sealed. A pressure equal or higher that atmospheric pressure is applied to the electrolyte vessel onto the sample injection end. The capillaries are provided. The sample injection end, said fluorescence detection positions and said sample elution end are arranged substantially at identical distances on one identical plane.
Yet, a further embodiment of the present invention is a capillary electrophoresis system comprising a capillary for separating a plurality of fluorescently-labeled samples by electrophoresis by applying a voltage across both ends of the capillary, means for introducing an electrophoretic separation medium into the capillary and for removing the electrophoretic separation medium from the capillary, an irradiation unit for irradiating a laser beam to the capillary and a detection unit for detecting fluorescence emitted from a plurality of samples that are electrophoretically separated.
A relationship is maintained between the geometrical characteristics of the capillary: T the height of a highest position of the capillary in a vertical direction, measured from a reference horizontal plane, E1 the height of the sample injection end in vertical direction from the reference horizontal plane, and L1 the capillary length between the sample injection end and the fluorescence detection position at which the laser beam is irradiated. The relationship is Txe2x88x92E1 greater than L1. The sample injection end points substantially vertically downward.
Another embodiment of the present invention is a capillary electrophoresis system comprising a capillary for containing a solution of an electrophoretic separation medium for separating fluorescently-labeled samples by electrophoresis by applying a voltage across both ends of the capillary, means for introducing the solution into the capillary and removing the solution from the capillary, an irradiation unit for irradiating a laser beam onto the capillary, a detection unit for detecting fluorescence emitted from a plurality of samples that are electrophoretically separated. The capillary has a fluorescence detection position where the laser beam is irradiated. The movements of the samples at the fluorescence detection position are substantially vertically upward.